The long-term goal is this research is to understand the molecular mechanism(s) permitting the emergence of new pathogenic viruses in humans, using influenza A viruses as a model. In the last three decades, several new viral pathogens appeared in humans after being maintained in small human populations or in other animal hosts. Pandemic influenza strains are classic examples of the latter type; the hemagglutinin (HA), neuraminidase (NA), and PBI genes of the 1957 and 1968 pandemic strains were derived from avian influenza viruses. Previous studies have demonstrated genetic changes in both the HA and NA of pandemic influenza strains that altered the host range of these viruses, although the structural basis of such effects are unknown. This proposal seeks to determine the molecular basis of influenza A virus host range restriction. Recent development of the reverse genetics techniques by Palese and colleagues, combined with information provided by the three- dimensional structures of the HA and NA, now permits direct examination of the relevance of specific amino acid alterations to the host range of influenza A viruses. The following specific aims are proposed: 1. Elucidation of the structural features of the HA that determine the host range specificity of influenza A viruses. Amino acid changes in the avian virus HA required for efficient replication of the virus in squirrel monkeys will be identified, in relation to its receptor specificity, by using reverse genetics to generate influenza viruses containing specific mutations in the HA. 2. Determination of the molecular features of the NA that influence the host range of influenza A viruses. The structural basis of the substrate specificity of the NA will be established by in vitro mutagenesis. The influence of the substrates specificity on the host range restriction of the virus will then be determined in both avian and mammalian systems by introducing specific mutations in the NA, using reverse genetics. Knowledge of the amino acid changes in the avian virus HA and NA required for efficient replication in mammals should allow us to assess the potential of avian- like viruses in nonhuman mammals to provide the genes for the generation of the pandemic strains, making it possible to predict (and possible prevent) future pandemic of human influenza. It could be also applied to the attenuation of human influenza viruses leading to the generation of live vaccine strains. Identification of the fine receptor and substrate specificities of the HA and NA should provide basic information needed for designing antiviral agents that would inhibit the activities of these viral constituents without affecting cellular processes.